牛分支杆菌CFP-10的原核表达及其在牛结核病诊断中的应用

低分子质量的蛋白抗原CFP-10是一种重要的牛分支杆菌早期分泌蛋白.为了检测该蛋白和其他几种抗原在牛结核病诊断中的临床应用,对CFP-10的基因进行克隆,鉴定,并在原核系统中表达.PCR法扩增cfp-10基因片段,连接到pET-22b( )原核表达载体中,再转入表达宿主E.coli BL21(DE3)PlysS菌株内,用IPTG诱导,进行蛋白表达、纯化.分别以CFP-10、ESAT-6、MPT83、MPT70、牛PPD、CFP-10与ESAT-6混合蛋白,MPT83与MPT70混合蛋白为抗原,用间接ELISA法诊断牛结核病.结果表明,以CFP-10与ESAT-6混合蛋白作为抗原检测牛结核病的特异性和敏感性分别达到了100%和63.6%,均超过了其他单抗原或抗原组合,为以筛选合适抗原为基础的血清学诊断技术提供了有力的支持并创造了良好的应用前景.

Prokaryotic Expression of CFP-10 Protein Secreted by Mycobacterium bovine and Its Application in the Diagnosis of Bovine Tuberculosis

The purpose of this study was to clone,identify and express the CFP-10 protein secreted by Mycobacterium bovis in prokaryotic expression vector.The cfp-10 gene fragment was firstly amplified by PCR and cloned to prokaryotic expression vector pET22b( ),and then the recombinant plasmid pET22b( )-cfp10 was transformed to E.coli BL21(DE3) PlysS strain and induced with IPTG to express the fusion protein.And by using the method of iELISA,CFP-10,ESAT-6,MPT83,MPT70,PPD-B,combination of CFP-10 and ESAT-6 and combination of MPT83 and MPT70 were tested for their potentials as diagnostic markers in diagnosing the bovine tuberculosis.The result showed that the combination of CFP-10 and ESAT-6 presented superior specificity and sensibility,reaching 100% and 63.6%,respectively,which surpasses any other single antigen or combination of different antigens.Such conclusion greatly supports the future perspective of diagnosing the bovine tuberculosis by testing the level of antibody released from the serum,which lies much on the effective selection of diverse antigens.